Atherosclerosis is a leading cause of death and morbidity in the US, and has complex, multifactorial pathophysiology. The goal of this exploratory grant is to determine whether intestinal colonization by specific commensal bacteria modulates T cell responses so as to promote atherosclerosis. The idea relies on key recent lines of evidence and our preliminary data. Colonization of the gut of mice by specific intestinal microbes promotes local differentiation of specific T cell subsets, including proinflammatory Th1 and Th17 cells, and anti-inflammatory regulatory T cells. Th17 cell differentiation within the gut is promoted by segmented filamentous bacteria (SFB), which are long Gram-positive spore-forming anaerobes that adhere tightly to small intestine epithelial cells. Because SFB promote Th17 differentiation, and Th17 cells are thought to exert an atherogenic role in apolipoprotein E-deficient (ApoE KO) mice, we hypothesize that intestinal SFB colonization increases susceptibility of ApoE KO mice to atherosclerosis. Our preliminary findings support this idea, because we found that serum IL-17 and splenic IL-17 mRNA levels were initially low in ApoE KO mice, but co-housing these mice with SFB-colonized mice led to their increased incidence of small intestinal SFB accompanied by increased levels of serum IL-17, splenic IL-17 mRNA, and splenocytic IL-17 release in vitro. Our working hypothesis is that colonization of the small intestine by SFB promotes development within the intestinal wall of IL-17-expressing T cells, which are then mobilized into the lymphatic system and recruited to sites of aortic inflammation, where they act locally by producing IL-17 and IFN-?, which synergistically promote atherogenesis. Based on preliminary findings that Toll-like receptor-4 (TLR4)-deficient mice have increased abundance of intestinal SFB, we also hypothesize that TLR4 signaling can limit intestinal SFB colonization in ApoE KO mice. The specific aims are to determine whether 1) colonization of the small intestine of ApoE KO mice by SFB drives local induction of small intestinal, systemic, and intralesional Th17 cell populations, and thereby promotes aortic lesion formation; and 2) TLR4-dependent intestinal expression of antibacterial proteins inhibits intestinal colonization with SFB in ApoE KO mice with low levels of SFB exposure. This exploratory project will test the novel concept that a specific bacterium that resides in the gut, previously thought to be non-pathogenic, promotes atherogenesis. Validation of this idea will potentially alter the drivers of atherosclerosis research. The project will establish and exploit a new interdisciplinary collaboration between established vascular (Dr. Beasley) and immunology (Dr. Huber) researchers at Tufts University. We will capitalize upon innovative and highly sensitive detection methods for early-stage atherogenesis and bacteria within a relevant animal model, to optimize long-term translational potential.